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Insect Biochem Mol Biol. 2014 Dec;55:61-9. doi: 10.1016/j.ibmb.2014.10.004. Epub 2014 Oct 30.

Re-examination of a α-chymotrypsin-solubilized laccase in the pupal cuticle of the silkworm, Bombyx mori: Insights into the regulation system for laccase activation during the ecdysis process.

Author information

1
Department of Biological Sciences, Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192-0397, Japan. Electronic address: asano-tsunaki@tmu.ac.jp.
2
Department of Chemistry, Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
3
Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA.
4
Department of Biological Sciences, Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192-0397, Japan.
5
Division of Cellular Biosignal Sciences, Department of Biochemistry, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.

Abstract

The laccase in the pupal cuticle of the silkworm, Bombyx mori, is thought to accumulate as an inactive precursor that can be activated stage-dependently. In this study we isolated an 81-kDa laccase from cuticular extract of B. mori that was prepared by digestion of the pupal cuticles with α-chymotrypsin. The mass spectrometric analysis of the purified protein indicates that this 81-kDa laccase is a product of the Bombyx laccase2 gene. The purified 81-kDa laccase (α-chymotrypsin-solubilized Bombyx laccase2: Bm-clac2) has an N-terminal sequence of RNPADS that corresponds to Arg146 to Ser151 of the deduced protein sequence of Bmlaccase2 cDNA, indicating that Bm-clac2 lacks the N-terminal part upstream from residue Arg146. Bm-clac2 shows enzymatic activity, but its specific activity is increased around 17-fold after treatment with trypsin, which involves cleavage of peptide bonds at the C-terminal region. We also found that the activity of Bm-clac2 is increased in the presence of isopropanol. In previous reports, proteolytic processing has been hypothesized as a system for laccase activation in vivo, but the present result implies that this type of processing is not the only way to convert Bm-clac2 to the high-activity enzyme.

KEYWORDS:

Activation; Laccase; Phylogenetic analysis

PMID:
25460512
DOI:
10.1016/j.ibmb.2014.10.004
[Indexed for MEDLINE]

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